Development of sequence characterized amplified region (SCAR) markers linked to race-specific resistance to Striga gesnerioides in cowpea (Vigna unguiculata L.)

An amplified fragement length polymorphism (AFLP) fragment, E-ACT/M-CAA524, tightly linked to the Striga gesnerioides race 1 (SG1) resistance gene Rsg-2-1 in cowpea (Vigna unguiculata L.) was isolated by polyacrylamide gel electrophoresis, cloned, and its nucleotide sequence determined. Based on the resulting sequence information, a pair of sequence specific primers were designed and used to isolate identical and similar fragments from cowpea genomic DNA of different cowpea lines by polymerase chain reaction (PCR) amplification. The primers amplified a ~500 bp fragment (SCAR marker designated as 61R) that was present in the resistant parent TVU14676, absent in susceptible parent IT84S-2246, and segregated with the resistance phenotype in an F2 population, derived from a cross of these two genotypes. The same primers were used to isolate a fragment similar to 61R from another S. gesnerioides resistant line Kvx 61-1. The sequence of this fragment was used to design a new combination of primers that developed a second SCAR marker, designated as 61R-M2. Subsequent analysis of the three markers, E-ACT/M-CAA524, 61R and 61M2 showed that they are linked to each other by 0.6 centimorgans (cM). The utility of these SCARs in marker assisted selection programs for cowpea was discussed.Keywords: Striga gesnerioides, centimorgans (cM), race specific resistance, amplified fragment length polymorphism (AFLP), sequence characterized amplified region (SCAR), marker assisted selection (MAS

CGKB: an annotation knowledge base for cowpea (Vigna unguiculata L.) methylation filtered genomic genespace sequences

<p>Abstract</p> <p>Background</p> <p>Cowpea [<it>Vigna unguiculata </it>(L.) Walp.] is one of the most important food and forage legumes in the semi-arid tropics because of its ability to tolerate drought and grow on poor soils. It is cultivated mostly by poor farmers in developing countries, with 80% of production taking place in the dry savannah of tropical West and Central Africa. Cowpea is largely an underexploited crop with relatively little genomic information available for use in applied plant breeding. The goal of the Cowpea Genomics Initiative (CGI), funded by the Kirkhouse Trust, a UK-based charitable organization, is to leverage modern molecular genetic tools for gene discovery and cowpea improvement. One aspect of the initiative is the sequencing of the gene-rich region of the cowpea genome (termed the genespace) recovered using methylation filtration technology and providing annotation and analysis of the sequence data.</p> <p>Description</p> <p>CGKB, Cowpea Genespace/Genomics Knowledge Base, is an annotation knowledge base developed under the CGI. The database is based on information derived from 298,848 cowpea genespace sequences (GSS) isolated by methylation filtering of genomic DNA. The CGKB consists of three knowledge bases: GSS annotation and comparative genomics knowledge base, GSS enzyme and metabolic pathway knowledge base, and GSS simple sequence repeats (SSRs) knowledge base for molecular marker discovery. A homology-based approach was applied for annotations of the GSS, mainly using BLASTX against four public FASTA formatted protein databases (NCBI GenBank Proteins, UniProtKB-Swiss-Prot, UniprotKB-PIR (Protein Information Resource), and UniProtKB-TrEMBL). Comparative genome analysis was done by BLASTX searches of the cowpea GSS against four plant proteomes from <it>Arabidopsis thaliana, Oryza sativa, Medicago truncatula</it>, and <it>Populus trichocarpa</it>. The possible exons and introns on each cowpea GSS were predicted using the HMM-based Genscan gene predication program and the potential domains on annotated GSS were analyzed using the HMMER package against the Pfam database. The annotated GSS were also assigned with Gene Ontology annotation terms and integrated with 228 curated plant metabolic pathways from the <it>Arabidopsis </it>Information Resource (TAIR) knowledge base. The UniProtKB-Swiss-Prot ENZYME database was used to assign putative enzymatic function to each GSS. Each GSS was also analyzed with the Tandem Repeat Finder (TRF) program in order to identify potential SSRs for molecular marker discovery. The raw sequence data, processed annotation, and SSR results were stored in relational tables designed in key-value pair fashion using a PostgreSQL relational database management system. The biological knowledge derived from the sequence data and processed results are represented as views or materialized views in the relational database management system. All materialized views are indexed for quick data access and retrieval. Data processing and analysis pipelines were implemented using the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The CPU intensive data processing and analysis pipelines were run on a computer cluster of more than 30 dual-processor Apple XServes. A job management system called Vela was created as a robust way to submit large numbers of jobs to the Portable Batch System (PBS).</p> <p>Conclusion</p> <p>CGKB is an integrated and annotated resource for cowpea GSS with features of homology-based and HMM-based annotations, enzyme and pathway annotations, GO term annotation, toolkits, and a large number of other facilities to perform complex queries. The cowpea GSS, chloroplast sequences, mitochondrial sequences, retroelements, and SSR sequences are available as FASTA formatted files and downloadable at CGKB. This database and web interface are publicly accessible at <url>http://cowpeagenomics.med.virginia.edu/CGKB/</url>.</p

TOBFAC: the database of tobacco transcription factors

<p>Abstract</p> <p>Background</p> <p>Regulation of gene expression at the level of transcription is a major control point in many biological processes. Transcription factors (TFs) can activate and/or repress the transcriptional rate of target genes and vascular plant genomes devote approximately 7% of their coding capacity to TFs. Global analysis of TFs has only been performed for three complete higher plant genomes – Arabidopsis (<it>Arabidopsis thaliana</it>), poplar (<it>Populus trichocarpa</it>) and rice (<it>Oryza sativa</it>). Presently, no large-scale analysis of TFs has been made from a member of the <it>Solanaceae</it>, one of the most important families of vascular plants. To fill this void, we have analysed tobacco (<it>Nicotiana tabacum</it>) TFs using a dataset of 1,159,022 gene-space sequence reads (GSRs) obtained by methylation filtering of the tobacco genome. An analytical pipeline was developed to isolate TF sequences from the GSR data set. This involved multiple (typically 10–15) independent searches with different versions of the TF family-defining domain(s) (normally the DNA-binding domain) followed by assembly into contigs and verification. Our analysis revealed that tobacco contains a minimum of 2,513 TFs representing all of the 64 well-characterised plant TF families. The number of TFs in tobacco is higher than previously reported for Arabidopsis and rice.</p> <p>Results</p> <p>TOBFAC: the database of tobacco transcription factors, is an integrative database that provides a portal to sequence and phylogeny data for the identified TFs, together with a large quantity of other data concerning TFs in tobacco. The database contains an individual page dedicated to each of the 64 TF families. These contain background information, domain architecture via Pfam links, a list of all sequences and an assessment of the minimum number of TFs in this family in tobacco. Downloadable phylogenetic trees of the major families are provided along with detailed information on the bioinformatic pipeline that was used to find all family members. TOBFAC also contains EST data, a list of published tobacco TFs and a list of papers concerning tobacco TFs. The sequences and annotation data are stored in relational tables using a PostgrelSQL relational database management system. The data processing and analysis pipelines used the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The computationally intensive data processing and analysis pipelines were run on an Apple XServe cluster with more than 20 nodes.</p> <p>Conclusion</p> <p>TOBFAC is an expandable knowledgebase of tobacco TFs with data currently available for over 2,513 TFs from 64 gene families. TOBFAC integrates available sequence information, phylogenetic analysis, and EST data with published reports on tobacco TF function. The database provides a major resource for the study of gene expression in tobacco and the <it>Solanaceae </it>and helps to fill a current gap in studies of TF families across the plant kingdom. TOBFAC is publicly accessible at <url>http://compsysbio.achs.virginia.edu/tobfac/</url>.</p

Sequencing and analysis of the gene-rich space of cowpea

<p>Abstract</p> <p>Background</p> <p>Cowpea, <it>Vigna unguiculata </it>(L.) Walp., is one of the most important food and forage legumes in the semi-arid tropics because of its drought tolerance and ability to grow on poor quality soils. Approximately 80% of cowpea production takes place in the dry savannahs of tropical West and Central Africa, mostly by poor subsistence farmers. Despite its economic and social importance in the developing world, cowpea remains to a large extent an underexploited crop. Among the major goals of cowpea breeding and improvement programs is the stacking of desirable agronomic traits, such as disease and pest resistance and response to abiotic stresses. Implementation of marker-assisted selection and breeding programs is severely limited by a paucity of trait-linked markers and a general lack of information on gene structure and organization. With a nuclear genome size estimated at ~620 Mb, the cowpea genome is an ideal target for reduced representation sequencing.</p> <p>Results</p> <p>We report here the sequencing and analysis of the gene-rich, hypomethylated portion of the cowpea genome selectively cloned by methylation filtration (MF) technology. Over 250,000 gene-space sequence reads (GSRs) with an average length of 610 bp were generated, yielding ~160 Mb of sequence information. The GSRs were assembled, annotated by BLAST homology searches of four public protein annotation databases and four plant proteomes (<it>A. thaliana</it>, <it>M. truncatula, O. sativa</it>, and <it>P. trichocarpa</it>), and analyzed using various domain and gene modeling tools. A total of 41,260 GSR assemblies and singletons were annotated, of which 19,786 have unique GenBank accession numbers. Within the GSR dataset, 29% of the sequences were annotated using the Arabidopsis Gene Ontology (GO) with the largest categories of assigned function being catalytic activity and metabolic processes, groups that include the majority of cellular enzymes and components of amino acid, carbohydrate and lipid metabolism. A total of 5,888 GSRs had homology to genes encoding transcription factors (TFs) and transcription associated factors (TAFs) representing about 5% of the total annotated sequences in the dataset. Sixty-two (62) of the 64 well-characterized plant transcription factor (TF) gene families are represented in the cowpea GSRs, and these families are of similar size and phylogenetic organization to those characterized in other plants. The cowpea GSRs also provides a rich source of genes involved in photoperiodic control, symbiosis, and defense-related responses. Comparisons to available databases revealed that about 74% of cowpea ESTs and 70% of all legume ESTs were represented in the GSR dataset. As approximately 12% of all GSRs contain an identifiable simple-sequence repeat, the dataset is a powerful resource for the design of microsatellite markers.</p> <p>Conclusion</p> <p>The availability of extensive publicly available genomic data for cowpea, a non-model legume with significant importance in the developing world, represents a significant step forward in legume research. Not only does the gene space sequence enable the detailed analysis of gene structure, gene family organization and phylogenetic relationships within cowpea, but it also facilitates the characterization of syntenic relationships with other cultivated and model legumes, and will contribute to determining patterns of chromosomal evolution in the Leguminosae. The micro and macrosyntenic relationships detected between cowpea and other cultivated and model legumes should simplify the identification of informative markers for marker-assisted trait selection and map-based gene isolation necessary for cowpea improvement.</p

Confirmation de QTL et validation de marqueurs SNPs associés à la résistance du niébé à Colletotrichum capsici, agent responsable de la maladie des taches brunes

Le niébé (Vigna unguiculata (L.) Walp.) est une légumineuse à graine très importante et constitue la principale source de protéines végétales pour l’alimentation des populations d’Afrique Subsaharienne. Sa production au Burkina Faso est entravée par la maladie des taches brunes provoquée par un champignon, Colletotrichum capsici (Syd.) Butler et Bisby. C’est dans la perspective d’accroître la productivité du niébé que nous avons entrepris de renforcer la lutte variétale contre cet agent pathogène. L’identification de marqueurs SNPs (Single Nucleotide Polymorphism) et QTL liés à la résistance à la maladie des taches brunes a été entrepris à partir d’une population biparentale F2 issus du croisement entre la variété sensible Tiligré et celle résistante KN-1. L’analyse QTL de la résistance du niébé à C. capsici à partir de la méthode ICIM add. a permis de confirmer et de valider respectivement un QTL majeur dénommé qBBDR2.1 et 9 marqueurs SNPs convertis, lesquels ont été cartographiés sur le chromosome Vu02 du niébé. Ce QTL dominant a présenté des effets additifs élevés liés aux allèles favorables de KN-1 et des valeurs de PVE de l’ordre de 51,50% et 55,33%, respectivement aux 21ème et 28ème JAI. English title: Confirmation of QTL mapping and validation of SNPs markers associated to cowpea resistance to Colletotrichum capsici, causal agent of brown blotch disease Cowpea (Vigna unguiculata (L.)Walp.) is one of the most important grain legume crops and constitutes the main source of plant protein for people food in sub-Saharan Africa. Cowpea production in Burkina Faso is constrained by brown blotch disease caused by a fungal,&nbsp; Colletotrichum capsici (Syd.) Butler and Bisby. In order to increase cowpea productivity we initiated a project to enhance host plant resistance to control the pathogen. The identification of SNP (Single Nucleotide Polymorphism) markers and QTL associated with brown blotch disease resistance was undertaken from a bi-parental F2 population resulting from a cross between the sensitive variety Tiligre and the resistant KN-1 to the disease. QTL analysis of cowpea resistance to C. capsici using the ICIM add method. Allowed to confirm and validate respectively a major QTL named qBBDR2.1 and 9 converted SNP markers, which were mapped on cowpea chromosome Vu02. This dominant QTL showed higher additive effects associated to alleles from KN-1 and PVE values of 51.50% and 55.33% respectively at 21 and 28 days after inoculatio

Genome Resources for Climate‐Resilient Cowpea, an Essential Crop for Food Security

Cowpea (Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought‐prone climates, and a primary source of protein in sub‐Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K‐499‐35 include a whole‐genome shotgun (WGS) assembly, a bacterial artificial chromosome (BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi‐parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited‐input small‐holder farming and climate stress

Functional genomics of a generalist parasitic plant: Laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression

Abstract Background Orobanchaceae is the only plant family with members representing the full range of parasitic lifestyles plus a free-living lineage sister to all parasitic lineages, Lindenbergia. A generalist member of this family, and an important parasitic plant model, Triphysaria versicolor regularly feeds upon a wide range of host plants. Here, we compare de novo assembled transcriptomes generated from laser micro-dissected tissues at the host-parasite interface to uncover details of the largely uncharacterized interaction between parasitic plants and their hosts. Results The interaction of Triphysaria with the distantly related hosts Zea mays and Medicago truncatula reveals dramatic host-specific gene expression patterns. Relative to above ground tissues, gene families are disproportionally represented at the interface including enrichment for transcription factors and genes of unknown function. Quantitative Real-Time PCR of a T. versicolor β-expansin shows strong differential (120x) upregulation in response to the monocot host Z. mays; a result that is concordant with our read count estimates. Pathogenesis-related proteins, other cell wall modifying enzymes, and orthologs of genes with unknown function (annotated as such in sequenced plant genomes) are among the parasite genes highly expressed by T. versicolor at the parasite-host interface. Conclusions Laser capture microdissection makes it possible to sample the small region of cells at the epicenter of parasite host interactions. The results of our analysis suggest that T. versicolor’s generalist strategy involves a reliance on overlapping but distinct gene sets, depending upon the host plant it is parasitizing. The massive upregulation of a T. versicolor β-expansin is suggestive of a mechanism for parasite success on grass hosts. In this preliminary study of the interface transcriptomes, we have shown that T. versicolor, and the Orobanchaceae in general, provide excellent opportunities for the characterization of plant genes with unknown functions

Parasitic Plants Striga and Phelipanche Dependent upon Exogenous Strigolactones for Germination Have Retained Genes for Strigolactone Biosynthesis

Abstract Strigolactones are plant hormones with multiple functions, including regulating various aspects of plant architecture such as shoot branching, facilitating the colonization of plant roots by arbuscular mycorrhizal fungi, and acting as seed germination stimulants for certain parasitic plants of the family Orobanchaceae. The obligate parasitic species Phelipanche aegyptiaca and Striga hermonthica require strigolactones for germination, while the facultative parasite Triphysaria versicolor does not. It has been hypothesized that P. aegyptiaca and S. hermonthica would have undergone evolutionary loss of strigolactone biosynthesis as a part of their mechanism to enable specific detection of exogenous strigolactones. We analyzed the transcriptomes of P. aegyptiaca, S. hermonthica and T. versicolor and identified genes known to act in strigolactone synthesis (D27, CCD7, CCD8, and MAX1), perception (MAX2 and D14) and transport (PDR12). These genes were then analyzed to assess likelihood of function. Transcripts of all strigolactone-related genes were found M. Das et al. 1152 in P. aegyptiaca and S. hermonthica, and evidence points to their encoding functional proteins. Gene open reading frames were consistent with homologs from Arabidopsis and other strigolactone-producing plants, and all genes were expressed in parasite tissues. In general, the genes related to strigolactone synthesis and perception appeared to be evolving under codon-based selective constraints in strigolactone-dependent species. Bioassays of S. hermonthica root extracts indicated the presence of strigolactone class stimulants on germination of P. aegyptiaca seeds. Taken together, these results indicate that Phelipanche aegyptiaca and S. hermonthica have retained functional genes involved in strigolactone biosynthesis, suggesting that the parasites use both endogenous and exogenous strigolactones and have mechanisms to differentiate the two